Preparation and isolation of 5′ capped mRNA

Abstract

The synthesis of capped/tagged RNA, methods of use and kits providing same are contemplated. Tagged RNA permits isolation of RNA transcripts in vitro. The ability to isolate and purify capped RNA results in improved transcription and translation and provides a tool for identifying RNA-protein interactions. Such capped RNA finds use in therapeutic applications, diagnosis and prognosis and in the treatment of cancers and HIV.

1. A composition comprising: 
       
         
           
           
               
               
           
         
         wherein,
 B is a nucleobase or a nucleobase having a linker attached thereto; 
 R 1  is selected from a halogen, OH, OCH 3 , and a linker; 
 R 2  is selected from H, OH, OCH 3 , and a linker; 
 R 3  is CH 3  or void; 
 R 4  is NH 2  or a linker; 
 R 5  is H or a linker; and 
 n is 1, 2 or 3; 
 and wherein at least one of B, R 1 , R 2 , R 4 , or R 5  comprises a linker and the linker is attached to a reporter moiety. 
 
       
     
     
       2. The composition as recited in  claim 1 , wherein said linker is selected from N, S, and O. 
     
     
       3. The composition as recited in  claim 1 , wherein said linker is selected from an aminoallyl ((—CH 2 ) n CH 2 NH 2 ) where n=2-18, a secondary amine and an alkyl (C 3 -C 10 )NH 2  chain. 
     
     
       4. The composition as recited in  claim 1 , wherein said reporter moiety is selected from an affinity tag and an epitope tag. 
     
     
       5. The composition as recited in  claim 4 , wherein said affinity tag is selected from biotin, iminobiotin, avidin, and streptavidin. 
     
     
       6. The composition as recited in  claim 5 , wherein said biotin is selected from C5-C20-biotin, SS-biotin, XX-biotin (6-((6-((biotinoyl)amino)hexanoyl)amino)hexanoic acid succinimidyl ester), and NHS ester compounds thereof. 
     
     
       7. The composition as recited in  claim 1  attached to a 5′ end of an RNA molecule. 
     
     
       8. The composition as recited in  claim 3  attached to the 5′ end of an RNA molecule. 
     
     
       9. The composition as recited in  claim 6  attached to the 5′ end of an RNA molecule. 
     
     
       10. The composition as recited in  claim 1 , wherein
 R 3  is void, B comprises a linker and the linker is attached to a reporter moiety. 
 
     
     
       11. The composition as recited in  claim 10 , wherein said reporter moiety is an affinity tag selected from biotin, iminobiotin, avidin, and streptavidin. 
     
     
       12. The composition as recited in  claim 10  attached to the 5′ end of an RNA molecule. 
     
     
       13. The composition as recited in  claim 1 , wherein
 R 3  is CH 3 , B comprises a linker and the linker is attached to a reporter moiety. 
 
     
     
       14. The composition as recited in  claim 13 , wherein said reporter moiety is an affinity tag selected from biotin, iminobiotin, avidin, and streptavidin. 
     
     
       15. The composition as recited in  claim 13  attached to the 5′ end of an RNA molecule. 
     
     
       16. The composition as recited in  claim 1 , wherein R 3  is CH 3 , one of R 1 , R 2 , R 4 , or R 5  comprises a linker and the linker is attached to a reporter moiety. 
     
     
       17. The composition as recited in  claim 16 , wherein said reporter moiety is an affinity tag selected from biotin, iminobiotin, avidin, and streptavidin. 
     
     
       18. The composition as recited in  claim 16  attached to the 5′ end of an RNA molecule. 
     
     
       19. A method for isolating a dinucleotide capped molecule comprising:
 a) providing a nucleic acid mixture comprising the composition as recited in  claim 7 ; 
 b) binding the reporter moiety of step a) to a substrate to form a complex; 
 c) extracting the complex of step b) from the nucleic acid mixture; 
 d) removing the linker from the capped nucleic acid; and 
 wherein capped nucleic acids are isolated. 
 
     
     
       20. A composition comprising:
 an antigen presenting cell transfected with the composition of  claim 7 . 
 
     
     
       21. A kit for capping an RNA transcript comprising a cap analog having the structure: 
       
         
           
           
               
               
           
         
         wherein,
 B is a nucleobase or a nucleobase having a linker attached thereto; 
 R 1  is selected from a halogen, OH, OCH 3 , and a linker; 
 R 2  is selected from OH, H, OCH 3 , and a linker; 
 R 3  is CH 3  or void; 
 R 4  is NH 2  or a linker; 
 R 5  is H or a linker; and 
 n is 1, 2 or 3; 
 and wherein at least one of B, R 1 , R 2 , R 4 , or R 5  comprises a linker; 
 a reporter moiety is attached to the linker; 
 at least R 1  or R 2  is OH; and 
 
         b) an RNA polymerase. 
       
     
     
       22. A cell comprising an RNA molecule wherein the composition of  claim 1  is attached to the RNA molecule.